The goal of this project is to define the molecular mechanisms involved in the replication of HIV and related retroviruses and to develop new strategies for AIDS therapy. Our research is currently focused on two broad areas of interest: reverse transcription and virus assembly. During reverse transcription, there are two strand transfer events that are required for synthesis of full-length plus- and minus-strand DNA copies of the viral RNA genome. This process is efficient only when the HIV-1 nucleocapsid protein (NC) is present. HIV-1 NC has two zinc fingers, each containing the invariant CCHC zinc-coordinating residues and other residues that form three short loops; only the sequence of the second loop is identical in both zinc fingers. A major question that we have been addressing concerns the functional significance of the two zinc fingers in HIV-1 NC. Recently, we reported that zinc coordination is required for the nucleic acid chaperone activity of NC, which catalyzes unfolding of the complex secondary structures in RNA and DNA strand transfer intermediates. We have now asked: (i) Can CCHH or CCCC, zinc binding motifs that are found in other zinc finger proteins, be substituted for CCHC; and (ii) Can the amino acid context surrounding the CCHC motifs be changed by exchanging or duplicating the zinc fingers? Our results demonstrate that for optimal strand transfer, the CCHC motifs cannot be replaced by CCHH or CCCC and the amino acid context in the loop regions must be preserved. We also find that the N-terminal zinc finger is a more critical determinant of NC nucleic acid chaperone activity than the C-terminal finger. Interestingly, comparison of our in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication. For studies on the role of the HIV-1 capsid protein (CA) in virus replication, we have made five additional viral mutants with alanine substitutions in conserved, hydrophobic N-terminal CA residues. All of these mutants have a distinct phenotype: virions produced are non-infectious, exhibit aberrant core morphology, and are unable to initiate viral DNA synthesis in the infected cell, although the particles contain a functional reverse transcriptase enzyme. Taken together, these results reveal the close connection between proper core morphology and the ability to undergo reverse transcription. Studies on the protein composition and stability of the mutant viral cores are now underway. A new project on the template and primer requirements for initiation of HIV-1 reverse transcription and the role of NC has been initiated.